PROJECT SUMMARY/ ABSTRACT The goal of this study is to identify the molecular mechanisms by which blueberry-derived microbial metabolites improve endothelial dysfunction during metabolic syndrome (MetS). We will employ multiomics approaches to determine the vascular effects of blueberry metabolites. MetS is an important risk factor for cardiovascular mortality and endothelial dysfunction plays a major role in the development of vascular complications. Hyperglycemia, dyslipidemia, and pro-inflammatory cytokines contribute to endothelial dysfunction in MetS. The vascular endothelium is covered with a glycocalyx which is comprised of proteoglycans [core proteins with glycosaminoglycans (GAG)]. Intact glycocalyx of healthy vasculature acts as a protective barrier and prevents endothelial dysfunction. Glycocalyx, importantly heparan sulfate proteoglycan (HSPG), is severely compromised in MetS. Hence, preservation and restoration of HSPG to improve endothelial dysfunction is a novel strategy to ameliorate vascular complications in MetS. Human studies support the vascular effects of blueberry anthocyanins. Anthocyanins are extensively metabolized by the gut microbiota in humans, suggesting their vascular benefits might be mediated by their microbial metabolites. Gut microbiota metabolize anthocyanins and anthocyanins support the growth of microbes indicating a two-way relationship between them. Our preliminary data show that :(i) blueberry supplementation improves vascular inflammation and dysfunction, and increases the beneficial bacteria in diabetic mice; (ii) key blueberry metabolites attenuate palmitate-induced endothelial inflammation and vascular dysfunction, and increase GAG production in endothelial cells (ECs) isolated from diabetic patients. However, studies are lacking that identify (i) the microbial metabolites of blueberries (Aim 1), (ii) the mechanisms by which blueberry-derived microbial metabolites improve endothelial dysfunction in MetS (Aim 2), and (iii) the most active metabolites responsible for the vascular effects of blueberries (Aim 3). Based on our preliminary studies, we hypothesize that blueberry attenuates endothelial dysfunction in MetS by improving HSPG and/or acting on multiple targets which is mediated through the microbial metabolites of blueberries. (1) Aim 1A: Determine the optimal dose of blueberry required to improve vascular inflammation and dysfunction in mice with MetS using two established models [diabetic db/db mice and high fat diet (HFD)-fed mice]. Aim 1B: Identify blueberry-derived `microbial metabolites'. (2) Aim 2: Determine the mechanisms by which blueberry-derived microbial metabolites improve endothelial dysfunction in MetS. (3) Aim 3A: Determine the impact of circulating metabolites on endothelial dysfunction. Aim 3B. Identify the most active microbial metabolite(s). We will use physiologically relevant models and state of the art techniques to evaluate the mechanistic roles of microbial metabolites of blueberries at the cellular level, tissue level and organism level. Our study will provide strong scientific rationale for recommending dietary intake of blueberries to improve vascular health in the US population and worldwide.